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CN1846785A - Freeze dried composition containing genetic engineering fusion protein - Google Patents

Freeze dried composition containing genetic engineering fusion protein Download PDF

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Publication number
CN1846785A
CN1846785A CN 200510063136 CN200510063136A CN1846785A CN 1846785 A CN1846785 A CN 1846785A CN 200510063136 CN200510063136 CN 200510063136 CN 200510063136 A CN200510063136 A CN 200510063136A CN 1846785 A CN1846785 A CN 1846785A
Authority
CN
China
Prior art keywords
ala
leu
gly
glu
arg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN 200510063136
Other languages
Chinese (zh)
Inventor
金美玉
马素永
聂李亚
文美玉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Northland Biotech Co Ltd
Original Assignee
Beijing Northland Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Northland Biotech Co Ltd filed Critical Beijing Northland Biotech Co Ltd
Priority to CN 200510063136 priority Critical patent/CN1846785A/en
Publication of CN1846785A publication Critical patent/CN1846785A/en
Pending legal-status Critical Current

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Abstract

The freeze dried composition containing engineered fusion protein has fusion protein of GnRH-PE and its derivatives, and main components including glycine and mannitol. In addition, the composition may also contain surfactant, amino acid and saccharide. The composition is freeze dried for preservation in long term without losing the bioactivity of the GnRH-PE and its derivatives.

Description

The freeze-dried composition that contains genetic engineering fusion protein
One. technical field
The invention belongs to the preservation field of biological product; biological product are the pharmaceutical grade protein of injection mostly; be difficult for stable the preservation; so often adopting freeze dried form preserves; the present invention mainly is a freeze drying protectant of selecting various combination for use, reaches protection GnRH-PE and the bioactive purpose of this class fusion rotein of derivant thereof.
Two. background technology
The freeze drying technology of biological product is exactly that the material that contains a large amount of moisture content is frozen into solid in advance; suitably heating under vacuum condition then makes steam directly distil from solid; and material itself is stayed in the ice shelf when freezing; therefore dried small product size is almost constant; the long preservation that helps albumen or other biological goods; in lyophilizing; selected freeze dried protective agent is crucial; albumin is as a kind of important protective agent; owing to can make sample that good molding is arranged after adding and to the active well protective effect of sample; for a long time often by people selected be conventional freeze drying protectant, as freeze drying protectant as genetic engineering interferon.But because getable human albumin product is blood products in the market; it is subjected to the potential danger of viral pollution bigger; no longer advocated at present and used albumin as the freeze drying protectant of biological product, so utilize novel freeze drying protectant just very important as the albumin succedaneum.
In the present invention, GnRH is meant human interstital-cell-stimulating hormone's releasing factor, what PE40 was meant false pseudomonas bacillus exotoxin A removes the remaining part of domain I, the C art end of GnRH and the N-terminal of PE40, perhaps the N-terminal of the C-terminal of PE40 and GnRH couples together by peptide bond, just formed the GnRH-PE fusion rotein, and various mutant.But this albuminoid is the guiding protein of a class specific killing adenocarcinoma class tumor, now confirm, adenocarcinoma class cell all can overexpression GnRH receptor, after guiding protein enters blood, GnRH by guiding can discern GnRH receptor that such tumor cell shows and can be specifically and receptors bind specifically, and coupled toxin moiety PE and derivant thereof can be killed this cell, thereby this fusion rotein can reach the purpose of kill tumor cell specifically, thereby reaches the purpose of treatment tumor.
GnRH-PE is the non-existent artificial protein of a class occurring in nature, so its stability is not strong.Test shows that GnRH-PE and derivant 4 degree thereof are placed 30 days artifact activity down and just begun to descend, and show that the freeze dried method of needs keeps its biological activity.The present invention has solved the instability problem of GnRH-PE and this albuminoid of derivant thereof by a series of experimental study, and it can be preserved easily, transportation.
Three. summary of the invention:
The present invention is by to GnRH-PE and derivant freeze-dried composition thereof with to the selection of pH; found the freeze-dried composition that is suitable for GnRH-PE and this class fusion rotein of derivant thereof; the albumin that such freeze-dried composition is habitually practised is safe, practical; and it is cheap; biological activity to GnRH-PE and derivant thereof has the better protect effect, for this class fusion rotein of mentioning among the present invention realizes that medicineization provides a good medicament platform.GnRH-PE and derivant thereof are as the possible effective medicine of a class to cancer, and its biological activity has embodied the effect of its specific killing tumor cell, so the present invention is at the positive effect that expection is arranged aspect the tumor cell treatment.
As GnRH-PE that uses among the present invention and derivant thereof, be the artificial protein that a class nature does not have, it is characterized by the fusion rotein that human interstital-cell-stimulating hormone's releasing hormone GnRH or its mutant and false pseudomonas bacillus exotoxin A (PE) or its mutant are formed, so can use a kind of albumen with aminoacid sequence shown in sequence 1 or the sequence 5, also can use and a kind of sequence 1 or sequence 5 aminoacid sequences be modified, as inserting, disappearance, the sequence of suddenling change certain or some aminoacid and obtaining is as long as keep its biological activity.In other words, also can adopt shown with sequence 1 and the sequence 5 substantially identical albumen of aminoacid of its aminoacid sequence, promptly except aminoacid sequence shown in sequence 1 and the sequence 5, comprise that also aminoacid sequence shown in sequence 1 and the sequence 5 is carried out part to be inserted, disappearance is suddenlyd change certain or some aminoacid and the sequence that obtains.Such as sequence 3 is to add a Met by the N-terminal in sequence 1, the 6th Gly is sported Ala, after the 10th amino acids of sequence 1, inserted the catenation sequence of His-Met-Ala-Glu-Glu, C-terminal in sequence 1 removes Arg-Glu-Asp-Leu-Lys, replaces with Lys-Asp-Glu-Leu and the sequence that obtains.It is worthy of note that the sequence that the present invention lists only is to further specify of the present invention, and do not constitute the await the reply restriction of claim the present invention.
The method that obtains GnRH-PE and derivant thereof can realize by the method for molecular cloning, the gene order of can be synthetic and can synthesizing destination protein in conjunction with the method for PCR by the full gene of multi-disc section, by means such as enzyme action connections gene is connected into the purpose carrier then, as pET etc., be transformed into corresponding host bacterium then, express.Express the back by the collection thalline, the broken host bacterium of methods such as Mechanical Method or osmotic pressure, by the chromatography method of routine, as ion-exchange chromatography, gel permeation chromatography and hydrophobic chromatography etc. are purified to destination protein.Bioactive mensuration to destination protein adopts mtt assay, at first the Hela cell is digested and collects, and cell is diluted to 6-8 * 10 with the RPMI RPMI-1640 4The cell suspension of individual/milliliter is inoculated in 96 orifice plates, the 100ul/ hole, and 37 degree were cultivated 4 hours.Contrast adds the 100ul culture fluid.Sample is diluted to 100ul/mL with the RPMI RPMI-1640 earlier, as starter hole, presses the dilute sample that concerns in 2.5 times in every hole and last hole then, each adds the every hole 100ul of sample of dilution, and 37 degree were cultivated 24 hours, take out,, measure and calculate IC with microplate reader 570nm with mtt assay dyeing 50Value.Lyophilizing procedure Selection-45 ℃ freezing 4 hours, evacuation then, programming rate be controlled at 2 degree/hour, after sample temperature surpasses 0 degree, heat up be controlled at 8 degree/hour, be up to 20 degree and kept 6 hours.Lyophilizing finishes.
Aspect the main protective agent of GnRH-PE and derivant thereof, tried out mannitol, glycine, the mixture of mannitol and glycine is using the concentration between the 3%-20% to find that it all has reasonable effect aspect lyophilizing skeleton forming.Lyophilizing has the phenomenon of subsiding when concentration is less than 3%, and considers the waste when protective agent concentration is high, and preferred protective agent concentration has been fixed on 5%.Aspect about pH, the pH of its freeze-dried composition is less than in 7.0 before lyophilizing, find after the sample lyophilizing and be not clear after the dissolving, but micro white, and decline has also appearred in the activity of sample, and the pH that considers finished product should not be greater than pH7.5, and we are chosen as 7.0-7.5 with the pH scope, preferred 7.25.
In addition, the protective agent in the freeze-dried composition can also contain at least a following material: monosaccharide or derivatives thereof, aminoacid, disaccharides, surfactant.Wherein the monosaccharide or derivatives thereof comprises glucose, fructose, sorbitol, dextran; Aminoacid comprises alanine, arginine, lysine; Disaccharides is a sucrose, lactose, trehalose, maltose; Surfactant comprises Tween-80 and Tween-20.
Three. the specific embodiment:
Below the example purpose be further to set forth the present invention, and can not constitute the await the reply restriction of claim of patent of the present invention.
Example one.
The prescription of freeze-dried composition 1 is, the 2mL dosage form, and the albumen 1mg of sequence 3, the main protective agent of lyophilizing is a mannitol, 100mg, pH are 7.25.Carry out lyophilizing.
Example two.
The prescription of freeze-dried composition 2 is, the 2mL dosage form, and the albumen 1mg of sequence 3, the main protective agent of lyophilizing is a mannitol, and 120mg also contains 0.004%Tween-80, and pH is 7.30.Carry out lyophilizing.
Example three.
The prescription of freeze-dried composition 3 is, the 2mL dosage form, and the albumen 1mg of sequence 4, the main protective agent of lyophilizing is a glycine, and 100mg also contains 0.004%Tween-80, and pH is 7.20.Carry out lyophilizing.
Example four.
The prescription of freeze-dried composition 4 is, the 1mL dosage form, and the albumen 1mg of sequence 5, the main protective agent of lyophilizing is mannitol 50mg, also contains 2% dextran, pH is 7.25.Carry out lyophilizing.
Example five.
The prescription of freeze-dried composition 5 is, the 2mL dosage form, and the albumen 1mg of sequence 6, the main protective agent of lyophilizing is a glycine, and 90mg also contains 3%Arg, and pH is 7.15.Carry out lyophilizing.
Example six.
The prescription of freeze-dried composition 6 is, the 2mL dosage form, and the albumen 1mg of sequence 2, the main protective agent of lyophilizing is glycine 50mg, mannitol 50mg, pH are 7.20.Carry out lyophilizing.
The result:
The freeze-dried composition sequence number IC before the lyophilizing 50 (ug/mL) IC after the lyophilizing 50(ug/mL) 37 degree are placed 7 days IC after the lyophilizing 50(ug/mL)
1 5.02 5.52 5.81
2 4.86 5.48 6.03
3 6.58 6.21 5.87
4 4.39 4.46 4.95
5 9.87 8.36 10.09
6 8.85 9.18 9.32
As can be seen from the above table, lyophilizing prescription of the present invention has been protected the biological activity of destination protein effectively.
Sequence table
＜110〉Beijing Nuo Silande Bioisystech Co., Ltd
＜120〉contain the freeze-dried composition of genetic engineering fusion protein
<140>
<141>
<160>6
<210>1
<211>371
<212>PRT
＜213〉sequence 1
<400>1
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Ser Leu Ala
1 5 10 15
Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe
20 25 30
Thr Arg His Arg Gln Pro Arg Gly Tro Glu Gln Leu Glu Gln Cys
35 40 45
Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg
50 55 60
Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala
65 70 75
Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln
80 85 90
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser
95 100 105
Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala
110 115 120
Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly
125 130 135
Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg
140 145 150
Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
155 160 165
Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu
170 175 180
Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val
185 190 195
Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe
200 205 210
Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg
215 220 225
Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala
230 235 240
Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala
245 250 255
Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr
260 265 270
Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val
275 280 285
Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile
290 295 300
Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly
305 310 315
Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro
320 325 330
Thr Asp Pro Asn Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile
335 340 345
Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
350 355 360
Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
365 370 371
<210>2
<211>361
<212>PRT
＜213〉sequence 2
<400>2
Met Glu His Trp Ser Tyr Gly Leu Arg Pro Gly His Met Ala Glu
1 5 10 15
Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
20 25 30
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
35 40 45
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
50 55 60
Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
65 70 75
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
80 85 90
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
95 100 105
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
110 115 120
Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp
125 130 135
Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly
140 145 150
Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp
155 160 165
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg
170 175 180
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala
185 190 195
Gln Ser Ile Val Phe Gly Gly Val Ala Ala Arg Ser Gln Asp Leu
200 205 210
Asp Ala Ile Trg Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu
215 220 225
Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg
230 235 240
Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser
245 250 255
Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
260 265 270
Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
275 280 285
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu
290 295 300
Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile
305 310 315
Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu
320 325 330
Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu
335 340 345
Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu
350 355 360
Lys
361
<210>3
<211>376
<212>PRT
＜213〉sequence 3
<400>3
Met Glu His Trp Ser Tyr Ala Leu Arg Pro Gly His Met Ala Glu
1 5 10 15
Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
20 25 30
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
35 40 45
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
50 55 60
Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
65 70 75
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
80 85 90
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
95 100 105
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
110 115 120
Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr
125 130 135
Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly
140 145 150
Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu
155 160 165
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
170 175 180
Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu
185 190 195
Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala
200 205 210
Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp
215 220 225
Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala
230 235 240
Leu Ala tyr Gly tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly
245 250 255
Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser
260 265 270
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
275 280 285
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro
290 295 300
Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg
305 310 315
Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val
320 325 330
Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp
335 340 345
Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala
350 355 360
Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu
365 370 375
Leu
376
<210>4
<211>360
<212>PRT
＜213〉sequence 4
<400>4
Met Glu His Trp Ser Tyr Ala Leu Arp Pro Gly His Met Ala Glu
1 5 10 15
Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
20 25 30
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
35 40 45
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
50 55 60
Leu Tvr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
65 70 75
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
80 85 90
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
95 100 105
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
110 115 120
Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp
125 130 135
Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly
140 145 150
Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp
155 160 165
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg
170 175 180
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala
185 190 195
Gln Ser Ile Val Phe Gly Gly Val Ala Ala Arg Ser Gln Asp Leu
200 205 210
Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu
215 220 225
Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg
230 235 240
Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser
245 250 255
Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
260 265 270
Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
275 280 285
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu
290 295 300
Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile
305 310 315
Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu
320 325 330
Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu
335 340 345
Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu Leu
350 355 360
<210>5
<211>348
<212>PRT
＜213〉sequence 5
<400>5
Met Gly Trp Glu Gln Leu Glu Gln Ser Gly Tyr Pro Val Gln Arg
1 5 10 15
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val
20 25 30
Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly
35 40 45
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu
50 55 60
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln
65 70 75
Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val
80 85 90
Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala
95 100 105
Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala
110 115 120
Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly
125 130 135
Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln
140 145 150
Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe
155 160 165
Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
170 175 180
Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
185 190 195
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp
200 205 210
Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val
215 220 225
Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu
230 235 240
Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His
245 250 255
Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu
260 265 270
Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg
275 280 285
Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val
290 295 300
Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala
305 310 315
Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Glu
320 325 330
His Trp Ser Tyr Ala Leu Arg Pro Gly Pro Gly Lys Pro Pro Lys
335 340 345
Asp Glu Leu
348
<210>6
<211>332
<212>PRT
＜213〉sequence 6
<400>6
Met Gly Ttp Glu Gln Leu Glu Gln Ser Gly Tyr Pro Val Gln Arg
1 5 10 15
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val
20 25 30
Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly
35 40 45
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu
50 55 60
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln
65 70 75
Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp
80 85 90
Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu
95 100 105
Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr
110 115 120
Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu
125 130 135
Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
140 145 150
Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser
155 160 165
Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp
170 175 180
Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
185 190 195
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro
200 205 210
Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala
215 220 225
Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro
230 235 240
Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly
245 250 255
Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr
260 265 270
Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly
275 280 285
Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile
290 295 300
Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Glu His
305 310 315
Trp Ser Tyr Ala Leu Arg Pro Gly Pro Gly Lys Pro Pro Lys Asp
320 325 330
Glu Leu
332

Claims (11)

1.一种含有基因工程融合蛋白的冻干组合物。其中的融合蛋白是指第一位氨基酸改为Glu的人促黄体激素释放激素GnRH或其突变体以及假单胞杆菌外毒素A(PE)或其突变体组成的融合蛋白。1. A freeze-dried composition containing genetic engineering fusion protein. The fusion protein refers to a fusion protein composed of human luteinizing hormone releasing hormone GnRH or its mutants whose first amino acid is changed to Glu and Pseudomonas exotoxin A (PE) or its mutants. 2.权利要求1中，GnRH的突变体可包括第六位氨基酸突变为Ala，Trp或Leu的分子。2. In claim 1, the GnRH mutant may include a molecule in which the sixth amino acid is mutated to Ala, Trp or Leu. 3.权利要求1中，PE的突变体可包括去掉1-252位氨基酸，剩下的253-613位氨基酸的一段，为PE40；可包括去掉1-252，365-380两段氨基酸序列，剩下的253-364和381-613两段氨基酸序列按原顺序相连，为PE38；可包括去掉PE分子的1-279位氨基酸，剩下280-613部分，并将287位Cys突变为Ser或Ala或Gly，为PE37；可包括去掉1-279和365-380两段氨基酸序列，剩下的280-364，381-613两段氨基酸序列按原顺序相连，并将287位的Cys突变为Ser或Ala或Gly，为PE35；可包括PE，PE40，PE38，PE37，PE35分子去掉C末端的5个氨基酸Arg-Glu-Asp-Leu-Lys，而替换成Lys-Asp-Glu-Leu的分子。3. In claim 1, the mutant of PE may include removing 1-252 amino acids, and a section of remaining 253-613 amino acids is PE40; it may include removing 1-252, 365-380 two amino acid sequences, and remaining The two amino acid sequences of 253-364 and 381-613 are connected according to the original sequence, which is PE38; it can include removing the 1-279 amino acids of the PE molecule, leaving the 280-613 part, and mutating the 287 Cys to Ser or Ala Or Gly, which is PE37; can include removing the two amino acid sequences of 1-279 and 365-380, the remaining two amino acid sequences of 280-364 and 381-613 are connected in the original order, and the Cys at position 287 is mutated to Ser or Ala or Gly is PE35; it can include PE, PE40, PE38, PE37, PE35 molecules to remove the 5 amino acids Arg-Glu-Asp-Leu-Lys at the C-terminus, and replace them with Lys-Asp-Glu-Leu molecules. 4.权利要求1中，冻干组合物中除了融合蛋白外，其冻干保护剂主要成分是甘露醇。4. In claim 1, in addition to the fusion protein in the lyophilized composition, the main component of the lyoprotectant is mannitol. 5.权利要求1中，冻干组合物中除了融合蛋白外，其冻干保护剂主要成分是甘氨酸。5. In claim 1, in addition to the fusion protein in the lyophilized composition, the main component of the lyoprotectant is glycine. 6.权利要求1中，冻干组合物中除了融合蛋白外，其冻干保护剂的主要成分是甘露醇和甘氨酸的混合物。6. In claim 1, in addition to the fusion protein in the lyophilized composition, the main component of the lyoprotectant is a mixture of mannitol and glycine. 7.权利要求4中，所用甘露醇的浓度是3％-20％，优选5％。7. In claim 4, the concentration of mannitol used is 3%-20%, preferably 5%. 8.权利要求5中，所用甘氨酸的浓度是3％-20％，优选5％。8. In claim 5, the concentration of glycine used is 3%-20%, preferably 5%. 9.权利要求6中，所用的甘露醇和甘氨酸的混合物的总浓度是3％-20％，优选5％。9. In claim 6, the total concentration of the mixture of mannitol and glycine used is 3%-20%, preferably 5%. 10.权利要求2、3、4、5、6中，冻干组合物的pH为7.0-7.5，优选7.25。10. In claims 2, 3, 4, 5 and 6, the pH of the freeze-dried composition is 7.0-7.5, preferably 7.25. 11.权利要求2、3、4、5、6中，冻干组合物中的保护剂还可以含有至少一种以下的物质：单糖或其衍生物，氨基酸类，二糖类，表面活性剂类。其中单糖或其衍生物包括葡萄糖，果糖，山梨醇，右旋糖酐；氨基酸类包括丙氨酸，精氨酸，赖氨酸；二糖类包括蔗糖，乳糖，海藻糖，麦芽糖；表面活性剂类包括Tween-80和Tween-20。11. In claim 2, 3, 4, 5, 6, the protective agent in the freeze-dried composition can also contain at least one of the following substances: monosaccharide or its derivatives, amino acids, disaccharides, surfactants kind. Among them, monosaccharides or their derivatives include glucose, fructose, sorbitol, and dextran; amino acids include alanine, arginine, and lysine; disaccharides include sucrose, lactose, trehalose, and maltose; surfactants include Tween-80 and Tween-20.
CN 200510063136 2005-04-05 2005-04-05 Freeze dried composition containing genetic engineering fusion protein Pending CN1846785A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101195822B (en) * 2006-12-08 2012-08-08 湖南康都制药有限公司 High affinity targeting fusion protein
CN102813933A (en) * 2012-08-30 2012-12-12 青岛康地恩药业股份有限公司 Chicken infectious bursal disease egg yolk antibody cryoprotectant
CN103864938A (en) * 2014-03-24 2014-06-18 北京博翱泰生物技术有限公司 Target-specificity double-mutant fused protein and preparation process thereof
CN110563835A (en) * 2018-06-05 2019-12-13 内蒙古伊利实业集团股份有限公司 Lactoferrin freeze-drying protective agent and application thereof
CN111195234A (en) * 2018-11-16 2020-05-26 鲁南制药集团股份有限公司 Recombinant FGF21-Fc fusion protein freeze-dried powder preparation

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101195822B (en) * 2006-12-08 2012-08-08 湖南康都制药有限公司 High affinity targeting fusion protein
CN102813933A (en) * 2012-08-30 2012-12-12 青岛康地恩药业股份有限公司 Chicken infectious bursal disease egg yolk antibody cryoprotectant
CN103864938A (en) * 2014-03-24 2014-06-18 北京博翱泰生物技术有限公司 Target-specificity double-mutant fused protein and preparation process thereof
CN110563835A (en) * 2018-06-05 2019-12-13 内蒙古伊利实业集团股份有限公司 Lactoferrin freeze-drying protective agent and application thereof
CN111195234A (en) * 2018-11-16 2020-05-26 鲁南制药集团股份有限公司 Recombinant FGF21-Fc fusion protein freeze-dried powder preparation
CN111195234B (en) * 2018-11-16 2022-08-26 鲁南制药集团股份有限公司 Recombinant FGF21-Fc fusion protein freeze-dried powder preparation

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